A method for increasing the fermentation yield of norvancomycin

Abstract

The present invention provides a method for improving the fermentation yield of norvancomycin, which belongs to the technical field of drug preparation, comprising inoculating oriental pseudomycotic acid bacteria into a seed culture medium to obtain a seed solution, inoculating the cultured seed solution into a fermentation culture medium for fermentation and culture, and adding a feed culture medium to the fermentation culture medium during the fermentation process; the feed culture medium comprises one or more of a trace element, a carbon source, a nitrogen source and a pH regulator, the trace element is potassium dihydrogen phosphate, the carbon source is one or two of glucose and sodium acetate, the nitrogen source is yeast powder, and the pH regulator is one or two of acetic acid and ammonia water. The method of the present invention can improve the concentration of the bacterial body in the fermentation process and improve the yield of norvancomycin.

Description

Technical Field

[0001] The invention relates to norvancomycin, in particular to a method for improving the fermentation yield of norvancomycin, belonging to the technical field of drug preparation. Background Art

[0002] Demethylvancomycin is a glycopeptide antibiotic independently developed and produced in my country. Its chemical structure, pharmacological properties and antibacterial effects are similar to those of vancomycin, but its treatment cost is lower than that of vancomycin. Its mechanism of action is to inhibit the synthesis of bacterial cell walls. It is mainly used for infections caused by Staphylococcus (methicillin-resistant strains), Enterococcus (ampicillin-resistant strains), Clostridium difficile, etc., such as endocarditis, sepsis, and pseudomembranous colitis. It is currently one of the first choice drugs for the treatment of penicillin-resistant bacteria infections and β-lactam antibiotic allergies.

[0003] At present, the domestic production of norvancomycin adopts the fermentation method of Pseudomonas orientalis. The components of the fermentation medium, culture conditions and feeding methods will have very complex effects on bacterial metabolism and metabolites. Due to different strains, or even if the same strain is bred after different company screening procedures, the characteristics of the strains will be different. This difference leads to certain differences in various production conditions such as temperature, ventilation, carbon source, nitrogen source, fermentation time, etc. during the fermentation production process. Although fed-batch fermentation has been widely used in the penicillin or vitamin fermentation industry, there is still no universally applicable feeding method in the fermentation production process of norvancomycin. Summary of the invention

[0004] In view of the above problems, the present invention provides a method for increasing the fermentation yield of norvancomycin, so as to increase the bacterial concentration in the fermentation process and increase the yield of norvancomycin.

[0005] To achieve the above object, the technical solution of the present invention is:

[0006] A method for increasing the fermentation yield of norvancomycin, comprising inoculating Amycolatopsis orientalis into a seed culture medium to obtain a seed solution, inoculating the cultured seed solution into a fermentation culture medium for fermentation, and adding a feed culture medium to the fermentation culture medium during the fermentation process;

[0007] The feed medium comprises one or more of trace elements, a carbon source, a nitrogen source and a pH regulator, wherein the trace element is potassium dihydrogen phosphate, the carbon source is one or both of glucose and sodium acetate, the nitrogen source is yeast powder, and the pH regulator is one or both of acetic acid and ammonia water.

[0008] Furthermore, the formula of the seed culture medium is: by mass percentage, 2.0 glucose, 0.5 starch, 0.5 yeast extract powder, 0.5 corn steep liquor powder, 0.5 calcium carbonate, 0.2 ammonium sulfate, 0.2 sodium chloride, 0.01 dipotassium hydrogen phosphate, and 0.2 corn oil.

[0009] Furthermore, the formula of the fermentation medium is as follows: by mass percentage, glucose 8.0, soybean powder 3.0, corn steep liquor dry powder 1.0, ammonium sulfate 0.5, calcium carbonate 0.5, sodium chloride 0.4, potassium dihydrogen phosphate 0.03, corn oil 0.3, and defoaming agent 0.06.

[0010] Furthermore, the fermentation culture cycle is 80-120 hours, the fermentation culture temperature is 30-32° C., the dissolved oxygen is more than 30%, and the pH is regulated to be 6.2-7.0.

[0011] Furthermore, during the fermentation process, potassium dihydrogen phosphate was added once or twice at 12h and 24h of fermentation, with the added amount being 0.3g per liter of fermentation liquid.

[0012] Furthermore, during the fermentation process, glucose is added according to the reducing sugar content from 24 hours of fermentation to 4 hours before tank release, the reducing sugar is detected every 4 hours during the fermentation process, and the glucose flow rate is adjusted to control the reducing sugar content at 30-60 g / L; after 24 hours of fermentation, sodium acetate is added at a rate of 0.01-0.02 g per liter of fermentation liquid per hour.

[0013] Furthermore, nitrogen source is added at the same time, with the amount of 0.1-0.3 g per liter of fermentation liquid per hour.

[0014] Furthermore, during the fermentation culture, the stirring speed is 200-500 rpm, the ventilation ratio is 1-2 vvm, and the stirring speed and the ventilation ratio cooperate to control the dissolved oxygen to be above 30%.

[0015] Furthermore, the seed solution is obtained by two steps: primary seed culture and secondary seed culture.

[0016] Primary seed culture: Take the frozen Amycolatopsis orientalis strain, thaw it at room temperature, apply it on the slant culture medium, and culture it at a constant temperature; rinse the slant spores with purified water, inoculate them in a seed culture shake bottle, and culture them at a constant temperature on a shaker to obtain the primary seed solution;

[0017] Secondary seed culture: take the primary seed solution and inoculate it into the seed tank, stir and culture to obtain the seed solution;

[0018] Add seed culture medium to seed culture shake flasks and seed tanks.

[0019] Furthermore, the formula of the slant culture medium is as follows: by mass ratio: 1.0 glucose, 1.0 soybean powder, 0.5 silkworm pupa powder, 0.5 sodium chloride, 0.03 calcium carbonate, and 1.0 agar powder.

[0020] The beneficial effects of the method for increasing the fermentation yield of norvancomycin of the present invention are:

[0021] The present invention provides a feed-batch culture method capable of increasing the yield of norvancomycin. Norvancomycin is a secondary metabolite produced by fermentation of Amycol. orientalis, and is generally not produced during the rapid cell growth period, but only when the bacterial growth reaches a relative static state. Therefore, the concentration that the bacterial body can reach in the early stage has a great influence on the production rate of the product in the middle and late stages. How to maintain high-speed metabolism after the bacterial growth reaches a plateau is closely related to the culture conditions and the feeding method. The existing feed-batch culture method of norvancomycin is not suitable for all strains, and a better culture method can achieve a higher product concentration, which is also the goal that production enterprises are constantly pursuing.

[0022] Phosphorus is an important element that is indispensable for forming cell membranes, but too high phosphorus will inhibit the generation of secondary metabolites. Therefore, the present invention only provides sufficient phosphorus during the rapid growth period of the thalli, which can increase the thalli concentration by more than 30%, thereby increasing the product concentration. Glucose can provide sufficient carbon sources for the thalli, and has less impact on the downstream product separation process than starch, corn oil, etc. The acetic acid group in sodium acetate participates in the tricarboxylic acid cycle in the biological sugar metabolism process, which can accelerate the metabolic rate of the thalli. Yeast powder is a standardized organic nitrogen source with a high protein content (protein accounts for more than 50% of the total weight of dry thalli), contains 18 kinds of amino acids, a full range of types, and rich in vitamins and minerals. The combination of sodium acetate and yeast powder can provide sufficient nutrients for the growth and metabolism of the thalli, significantly improve the product level, and the content of norvancomycin in the fermentation broth can reach more than 21g / L. DETAILED DESCRIPTION

[0023] The technical solutions in the embodiments of the present invention are described clearly and completely below. In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention can also be implemented in other ways different from those described herein, and those skilled in the art can make similar generalizations without violating the connotation of the present invention, so the present invention is not limited by the specific embodiments disclosed below.

[0024] A method for increasing the fermentation yield of norvancomycin, the scheme may specifically be:

[0025] Oriental pseudomycotic acid bacteria were inoculated into a seed culture medium to obtain a seed solution, and the cultured seed solution was inoculated into a fermentation culture medium for fermentation. The fermentation cycle was 80-120 hours, the fermentation temperature was 30-32°C, the dissolved oxygen was more than 30%, and the pH was regulated to be 6.2-7.0. During the fermentation for 12 hours and 24 hours, 0.3 g of potassium dihydrogen phosphate was added per liter of fermentation liquid. During the fermentation period from 24 hours to 4 hours before tank release, glucose was added according to the reducing sugar content. The reducing sugar was detected every 4 hours during the fermentation process, and the glucose flow rate was adjusted to control the reducing sugar content at 30-60 g / L; after 24 hours of fermentation, sodium acetate was added at 0.01-0.02 g per liter of fermentation liquid per hour; and nitrogen source was added at the same time, and the addition amount was 0.1-0.3 g per liter of fermentation liquid per hour.

[0026] The seed solution is obtained by two steps: primary seed culture and secondary seed culture, as follows:

[0027] Primary seed culture: Take the frozen strain, thaw it at room temperature, spread it on the slant culture medium, and culture it at 30℃ for 7 days. Rinse the slant spores with purified water, inoculate them in a 500mL seed culture shake flask at 1% inoculation, and culture it at 200rpm and 30℃ for 48h.

[0028] Secondary seed culture: Take the primary seed solution and inoculate it in a 5L seed tank with a 1% inoculation amount, temperature 30°C, stirring 400rpm, ventilation 1.5vvm, tank pressure 0.03MPa, and culture for 24h.

[0029] Slant culture medium formula (mass ratio %): glucose 1.0, soybean powder 1.0, silkworm pupa powder 0.5, sodium chloride 0.5, calcium carbonate 0.03, agar powder 1.0.

[0030] Seed culture medium formula (mass ratio %): glucose 2.0, starch 0.5, yeast extract powder 0.5, corn steep liquor dry powder 0.5, calcium carbonate 0.5, ammonium sulfate 0.2, sodium chloride 0.2, dipotassium hydrogen phosphate 0.01, corn oil 0.2.

[0031] Furthermore, the fermentation medium formula is (mass ratio %): glucose 8.0, soybean powder 3.0, corn steep liquor dry powder 1.0, ammonium sulfate 0.5, calcium carbonate 0.5, sodium chloride 0.4, potassium dihydrogen phosphate 0.03, corn oil 0.3, defoamer 0.06. After mixing and dissolving, the volume is fixed, sterilized at 120°C for 30 minutes, cooled to 31°C and adjusted to pH 7.00. During the fermentation culture, the stirring speed is 200-500rpm, the ventilation ratio is 1-2vvm, and the two cooperate to control the dissolved oxygen to be above 30%.

[0032] The amount of mycelium is determined by wet weight method, that is, take the fermentation liquid of mass M1 and place it in a graduated centrifuge tube, centrifuge it at 10000G for 10 minutes, pour out the supernatant, measure the mass of the precipitate M2, and the bacterial concentration is M2 / M1*100%. The concentration of reducing sugar and product is detected in the centrifuged supernatant.

[0033] The HPLC chromatographic detection conditions are: chromatographic column (C18, 4.6×250 mm, 5 μm), mobile phase (0.2% triethylamine aqueous solution at pH 3.2: acetonitrile: tetrahydrofuran = 93:6:1), column temperature 30°C, flow rate 1.0 mL / min, 20 μL injection, and detection at 280 nm wavelength.

[0034] Example 1 pH Control

[0035] A method for increasing the fermentation yield of norvancomycin, the specific implementation process is: a strain of oriental pseudomycotic acid bacteria is activated, after slant culture, primary seed culture and secondary seed culture, it is connected to a fermentation tank for fermentation, and a feed medium is added during the process to adjust the pH. The fermentation broth is centrifuged at 10000G for 10 minutes, and the supernatant is taken to detect the reducing sugar and product concentrations. The fermentation broth HPLC chromatographic detection conditions are: chromatographic column (C18, 4.6×250mm, 5μm), mobile phase (0.2% triethylamine aqueous solution at pH 3.2: acetonitrile: tetrahydrofuran = 93:6:1), column temperature 30°C, flow rate 1.0mL / min, injection 20μL, and detection at 280nm wavelength.

[0036] Primary seed culture: Take the frozen strain, thaw it at room temperature, spread it on the slant culture medium, and culture it at 30℃ for 7 days. Rinse the slant spores with purified water, inoculate them in a 500mL seed culture shake flask at 1% inoculation, and culture it at 200rpm and 30℃ for 48h.

[0037] Secondary seed culture: Take the primary seed solution and inoculate it in a 5L seed tank with a 1% inoculation amount, temperature 30°C, stirring 400rpm, ventilation 1.5vvm, tank pressure 0.03MPa, and culture for 24h.

[0038] Slant culture medium formula (mass ratio %): glucose 1.0, soybean powder 1.0, silkworm pupa powder 0.5, sodium chloride 0.5, calcium carbonate 0.03, agar powder 1.0.

[0039] Seed culture medium formula (mass ratio %): glucose 2.0, starch 0.5, yeast extract powder 0.5, corn steep liquor dry powder 0.5, calcium carbonate 0.5, ammonium sulfate 0.2, sodium chloride 0.2, dipotassium hydrogen phosphate 0.01, corn oil 0.2.

[0040] Preparation of fermentation medium: weigh 80g / L glucose, 30g / L soybean powder, 10g / L corn steep liquor powder, 5g / L ammonium sulfate, 5g / L calcium carbonate, 4g / L sodium chloride, 0.3g / L potassium dihydrogen phosphate, 3g / L corn oil, and 0.6g / L defoamer. Mix and dissolve, and dilute to 30L in a 50L tank. Sterilize at 120℃ for 30min, cool to 31℃ and adjust pH to 7.00.

[0041] Inoculation culture: Add 10% inoculation amount of norvancomycin secondary seed liquid to the fermentation tank, culture at 31°C for 80-120 hours, and adjust the dissolved oxygen to not less than 30% by ventilation and stirring. Adjust the pH to 6.2, 6.4, 6.6, 6.8, and 7.0 with acetic acid and ammonia water respectively. After 24 hours of fermentation, add glucose to control the reducing sugar content to 30-60g / L.

[0042] The specific results are shown in Table 1.

[0043] Example 2 pH Control and Phosphorus Source Addition

[0044] A method for increasing the fermentation yield of norvancomycin, the basic steps are the same as those in Example 1, specifically as follows:

[0045] (1) The fermentation medium was prepared in the same manner as in Example 1.

[0046] (2) Inoculation culture: The secondary seed solution of norvancomycin was inoculated with 10% of the seed solution and cultured at 31°C for 80-120 hours. The dissolved oxygen was regulated to be not less than 30% by ventilation and stirring. The pH was regulated to 6.4-6.6 by acetic acid and ammonia. After 24 hours, glucose was added to control the reducing sugar content to 30-60 g / L.

[0047] (3) Fed-batch culture: prepare a 10% potassium dihydrogen phosphate aqueous solution and add 90 mL at 12 h, 90 mL at 24 h, and 90 mL at 12 h and 24 h respectively.

[0048] The specific results are shown in Table 1.

[0049] Example 3: pH control, addition of phosphorus source and carbon source

[0050] A method for increasing the fermentation yield of norvancomycin, the basic steps are the same as those in Example 2, specifically as follows:

[0051] (1) The fermentation medium was prepared in the same manner as in Example 1.

[0052] (2) Inoculation and culture were the same as in Example 2.

[0053] (3) Supplementation of phosphorus source: prepare 10% potassium dihydrogen phosphate aqueous solution and add 90 mL each after 12 h and 24 h.

[0054] (4) Supplementation of carbon source: prepare 20%, 30%, and 40% sodium acetate aqueous solutions, and start adding sodium acetate solution after 24 hours of cultivation at a flow rate of 1.5 ml / h.

[0055] The specific results are shown in Table 1.

[0056] Example 4: pH adjustment, addition of phosphorus source, carbon source, and nitrogen source

[0057] A method for increasing the fermentation yield of norvancomycin, the basic steps are the same as those in Example 3, specifically as follows:

[0058] (1) The fermentation medium was prepared in the same manner as in Example 1.

[0059] (2) Inoculation and culture were the same as in Example 2.

[0060] (3) Adding phosphorus source is the same as in Example 3.

[0061] (4) Supplementation of carbon source: Prepare 30% sodium acetate aqueous solution and start adding sodium acetate solution after 24 h of cultivation at a flow rate of 1.5 ml / h.

[0062] (5) Adding nitrogen source: prepare 10% yeast powder aqueous solution, start feeding after 24 hours, the flow rate is 30mL / h, 60mL / h, 90mL / h respectively, and stop feeding 8 hours before the end of fermentation.

[0063] Table 1 Results of bacterial concentration and yield test in Examples 1-4

[0064]

[0065] From the results in Table 1, it can be seen that the optimum pH for fermentation of this strain is 6.4-6.6. Adding potassium dihydrogen phosphate at 0.3g per liter of fermentation liquid at 12h and 24h of fermentation will increase the bacterial concentration by more than 22% and the fermentation unit by more than 25%. After 24h, adding 0.015g of sodium acetate per liter of fermentation liquid per hour will increase the fermentation unit by more than 8%. After 24h, adding 0.2g of yeast powder per liter of fermentation liquid per hour will increase the fermentation unit by more than 16%.

[0066] Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

Claims

1. A method for increasing the fermentation yield of norvancomycin, characterized in that: inoculating Amycolatopsis orientalis into a seed culture medium to obtain a seed solution, inoculating the cultured seed solution into a fermentation culture medium for fermentation, and adding a feed culture medium to the fermentation culture medium during the fermentation process; The feed medium comprises one or more of trace elements, a carbon source, a nitrogen source and a pH regulator, wherein the trace element is potassium dihydrogen phosphate, the carbon source is one or both of glucose and sodium acetate, the nitrogen source is yeast powder, and the pH regulator is one or both of acetic acid and ammonia water.

2. The method for increasing the fermentation yield of norvancomycin according to claim 1, characterized in that: The formula of the seed culture medium is as follows: by mass ratio: 2.0 glucose, 0.5 starch, 0.5 yeast extract powder, 0.5 corn steep liquor powder, 0.5 calcium carbonate, 0.2 ammonium sulfate, 0.2 sodium chloride, 0.01 dipotassium hydrogen phosphate, and 0.2 corn oil.

3. The method for increasing the fermentation yield of norvancomycin according to claim 1, characterized in that: The formula of the fermentation medium is as follows: by mass percentage, glucose 8.0, soybean powder 3.0, corn steep liquor dry powder 1.0, ammonium sulfate 0.5, calcium carbonate 0.5, sodium chloride 0.4, potassium dihydrogen phosphate 0.03, corn oil 0.3, and defoaming agent 0.

06.

4. The method for increasing the fermentation yield of norvancomycin according to claim 1, characterized in that: The fermentation culture cycle is 80-120 hours, the fermentation culture temperature is 30-32° C., the dissolved oxygen is more than 30%, and the pH is regulated to be 6.2-7.

0.

5. The method for increasing the fermentation yield of norvancomycin according to claim 1, characterized in that: During the fermentation process, potassium dihydrogen phosphate was added once or twice at 12h and 24h of fermentation, with the addition amount being 0.3g per liter of fermentation liquid.

6. A method for increasing the fermentation yield of norvancomycin according to claim 5, characterized in that: During the fermentation process, glucose is added according to the reducing sugar content from 24 hours of fermentation to 4 hours before tank release. The reducing sugar is tested every 4 hours during the fermentation process, and the glucose flow rate is adjusted to control the reducing sugar content at 30-60g / L; after 24 hours of fermentation, sodium acetate is added at a rate of 0.01-0.02g per liter of fermentation liquid per hour.

7. A method for increasing the fermentation yield of norvancomycin according to claim 6, characterized in that: At the same time, nitrogen source is added in an amount of 0.1-0.3 g per liter of fermentation liquid per hour.

8. The method for increasing the fermentation yield of norvancomycin according to claim 1, characterized in that: During the fermentation process, the stirring speed is 200-500rpm, the ventilation ratio is 1-2vvm, and the stirring speed and the ventilation ratio cooperate to control the dissolved oxygen to be above 30%.

9. The method for increasing the fermentation yield of norvancomycin according to claim 1, characterized in that: The acquisition of seed solution includes two steps: primary seed culture and secondary seed culture. Primary seed culture: Take the frozen Amycolatopsis orientalis strain, thaw it at room temperature, apply it on the slant culture medium, and culture it at a constant temperature; rinse the slant spores with purified water, inoculate them in a seed culture shake bottle, and culture them at a constant temperature on a shaker to obtain the primary seed solution; Secondary seed culture: take the primary seed solution and inoculate it into the seed tank, stir and culture to obtain the seed solution; Add seed culture medium to seed culture shake flasks and seed tanks.

10. A method for increasing the fermentation yield of norvancomycin according to claim 9, characterized in that: The formula of the slant culture medium is as follows: by mass ratio, glucose 1.0, soybean powder 1.0, silkworm pupa powder 0.5, sodium chloride 0.5, calcium carbonate 0.03, and agar powder 1.0.

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